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基于Nrf2/HO-1信号通路的夜关门提取物对谷氨酸诱导小鼠海马细胞HT22损伤的保护作用及机制研究
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篇名: 基于Nrf2/HO-1信号通路的夜关门提取物对谷氨酸诱导小鼠海马细胞HT22损伤的保护作用及机制研究
TITLE: Study on Protective Effects and Mechanism of Lespedeza cuneata Extracts on Glutamate-induced Hippocampal Cells HT 22 Injury of Mice Based on Nrf 2/HO-1 Signaling Pathway
摘要: 目的:基于核因子2(Nrf2)/血红素加氧酶1(HO-1)信号通路研究夜关门提取物对谷氨酸诱导小鼠海马细胞HT22损伤的保护作用及机制。方法:采用谷氨酸(5mmol/L)建立HT22细胞损伤模型后,以水溶性维生素E为阳性对照(50?mol/L),采用MTT法检测0(空白对照)、25、50、100?g/mL夜关门的石油醚、二氯甲烷、乙酸乙酯提取物预处理12h后对谷氨酸诱导损伤细胞增殖的影响。以水溶性维生素E为阳性对照(50?mol/L),采用2′,7′-二氯荧光黄双乙酸盐探针(DCFH-DA)法检测0(空白对照)、25、50、100?g/mL夜关门二氯甲烷提取物预处理12h后对谷氨酸诱导损伤细胞中活性氧(ROS)水平的影响;以HO-1激动剂钴-原卟啉为阳性对照,采用Westernblotting法检测0(空白对照)、25、50、100?g/mL夜关门二氯甲烷提取物处理24h后对细胞中HO-1蛋白表达的影响;分别采用Westernblotting法(药物分别处理0.5、1、1.5h)和免疫荧光染色法(药物处理1h)检测100?g/mL夜关门二氯甲烷提取物处理后对细胞核内外Nrf2蛋白表达的影响。采用小干扰RNA(si-RNA)转染技术进行HO-1基因沉默后,考察100?g/mL夜关门二氯甲烷提取物对谷氨酸诱导损伤细胞的存活率以及细胞中ROS水平的影响。结果:与空白对照比较,50、100?g/mL夜关门二氯甲烷提取物均可显著升高细胞的存活率(P<0.05),并降低细胞中ROS水平(P<0.05);25、50、100?g/mL夜关门二氯甲烷提取物均可显著升高细胞中HO-1蛋白表达水平(P<0.05),100?g/mL夜关门二氯甲烷提取物可显著降低细胞质中Nrf2蛋白水平并升高细胞核中Nrf2蛋白水平(P<0.05)。HO-1基因沉默后,夜关门二氯甲烷提取物对谷氨酸诱导损伤细胞的促增殖以及降低ROS水平的作用被逆转(P<0.05)。结论:夜关门二氯甲烷提取物可通过激活Nrf2信号通路,诱导HO-1蛋白的表达,从而发挥对谷氨酸诱导损伤HT22细胞的保护作用。
ABSTRACT: OBJECTIVE:To study the protective effects of Lespedeza cunea ta extract on glutamate-induced hippocampal cells HT22 injury of mice and its possible mechanism based on Nrf 2/HO-1 signaling pathway. METHODS :Using glutamate (5 mmol/L) to extablish the injury model of HT 22 cells. Using water soluble vitamin E as positive control (50 ?mol/L),MTT assay was used to detect the effects of 0(blank control ),25,50,100 ?g/mL petroleum ether extract ,dichloromethane extract ,ethyl acetate extract of L. cuneata on the proliferation of glutamate-induced injury cellsafter pretreated for 12 h. Using water soluble vitamin E as positive control (50 ?mol/L),DCFH-DA assay was used to detect the effects of 0(blank control ),25,50,100 ?g/mL L. cuneata dichloromethane extract on the level of active oxygen (ROS)in glutamate-induced injury cells after pretreated with 12 h. Using HO-1 agonist CoPP as positive control ,Western blotting method was used to detect the effects of 0(blank control ),25,50,100 ?g/mL L. cuneata dichloromethane extract on the protein expression of HO- 1 after treated for 24 h. Western blotting method (treated for 0.5,1,1.5 h)and immunofluorescence staining (treated for 1 h)were used to detect the effects of 100 ?g/mL L. cuneata dichloromethane extract on protein expression of Nrf 2 inside and outside the nucleus. After HO-1 gene was silenced by small interfering RNA (Si RNA )transfection technology ,the effects of 100 ?g/mL L. cuneata dichloromethane extract on the survival rates of glutamate-induced injury cells and the level of ROS were detected. RESULTS :Compared with blank control ,50, 100 ?g/mL L. cuneata dichloromethane extract could significantly improve the survival rate of glutamate-induced injury cells (P< 0.05),while reduced the level of ROS (P<0.05). 25,50, 100 ?g/mL L. cuneata dichloromethane extract could increase the protein expression of HO- 1 in cells(P<0.05),while 100 com ?g/mL L. cuneata dichloromethane extract could significantly decrease the protein le vel of Nrf 2 in cytoplasm and increasethat in nucleus (P<0.05). After HO-1 gene silencing ,the effects of L. cuneata dichloromethane extract on the proliferation promotion of glutamate-induced injury cells and the reduction of ROS level were reversed (P<0.05). CONCLUSIONS :L. cuneata dichloromethane extract can protect HT 22 cells against injury induced by glutamate through activating Nrf 2 pathway,inducing HO- 1 expression.
期刊: 2020年第31卷第11期
编辑: 郭丰,黄山,李斌
AUTHORS: GUO Feng,HUANG Shan,LI Bin
关键字: 夜关门;谷氨酸;血红素加氧酶 1;核因子2;小鼠;海马细胞HT22;机制
KEYWORDS: Lespedeza cuneata ;Glutamate;HO-1;Nrf2;Mice;Hippocampal cells HT 22;Mechanism
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